熏马肠中生物胺氧化酶菌株的筛选与鉴定

为获得良好的产生物胺氧化酶的肉品发酵剂,本试验以新疆不同地区的44种熏马肠样品为研究对象,对产生物胺氧化酶菌进行筛选和鉴定,利用单层培养、双层显色的方法剔除产生物胺菌株,对分离后得到的菌株进行氧化酶试验、抑菌试验、耐盐和耐亚硝酸盐试验。采用DGGE剔除重复菌株,通过HPLC对所筛选菌株氧化生物胺能力进行验证,最后对筛选出的菌株提取DNA,并对其16S rDNA的V6-V8区段进行PCR扩增后测序。结果表明:本次试验共筛选出6株菌有氧化酶特性,耐盐能力在6%以上,耐亚硝酸盐能力在0.15 g/kg以上,对致病性大肠杆菌,单增李斯特菌,金黄色葡萄球菌三种致病菌有良好的抑制效果,经HPLC验证后氧化生物胺能力较强,分别为鼠李糖乳杆菌(Lactobacillus rhamnosus),枯草芽孢杆菌(Bacillus subtilis),腐生葡萄球菌(Staphylococcus saprophyticus),木糖葡萄球菌(Staphylococcus xylosus),戊糖片球菌(Pediococcus pentosaceus),植物乳杆菌(Lactobacillus plantarum)。为发酵肉制品的工业化生产和安全性提供了良好的理论依据。     

发酵剂对熏马肠成熟过程中生物胺含量变化的影响

利用从熏马肠中分离出的具有抑菌作用并可以产生物胺氧化酶的菌株,1株表皮葡萄球菌(Staphylococcus epidermidis),3株模仿葡萄球菌(Staphylococcus simulans)作为发酵剂加入到熏马肠,在熏马肠成熟过程中采用高效液相色谱检测不同发酵剂对生物胺含量变化的影响,进而阐明发酵剂对熏马肠中生物胺累积的控制作用。从研究结果可以看出,在马肠成熟过程中,B组对色胺、苯乙胺、腐胺、组胺、酪胺都有较显著的减少作用;C组对色胺、腐胺、尸胺、组胺、酪胺减少作用显著;D组对色胺、苯乙胺、腐胺、组胺、酪胺的减少作用显著;E组对组胺、苯乙胺、色胺的减少作用显著。添加发酵剂组对精胺、亚精胺的减少作用均不显著。结果表明,上述菌种对熏马肠成熟过程中生物胺产生有较好的抑制作用,具有肉品发酵剂的良好特性,为熏马肠以及其他发酵肉制品的工业化生产和安全性奠定了基础。     

熏马肠成熟过程中产生物胺氧化酶菌对生物胺作用的研究

该研究以从新疆熏马肠中筛选出来的6株产生物胺氧化酶菌株(鼠李糖乳杆菌、枯草芽孢杆、腐生葡萄球菌、木糖葡萄球菌、戊糖片球菌、植物乳杆菌)作为发酵剂加入熏马肠中,采用PCR-DGGE技术监测菌落动态变化,高效液相色谱法检测熏马肠中生物胺(色胺、腐胺、苯乙胺、组胺、酪胺、尸胺、精胺、亚精胺)含量变化。结果表明,在熏马肠成熟过程中发酵剂对生物胺有一定的抑制作用。其中以腐生葡萄球菌对色胺、腐生葡萄球菌对苯乙胺、木糖葡萄球菌对尸胺、鼠李糖乳杆菌对腐胺、腐生葡萄球菌对组胺和枯草芽孢杆菌对酪胺的抑制作用最为显著,减少程度分别为45.49%、57.54%、40.75%、51.03%、49.28%、64.33%。本研究为熏马肠以及其他发酵肉制品的提高安全性奠定了基础。     

乳酸菌中生物胺氧化酶菌株的筛选

为获得良好的具有生物胺降解能力的肉品发酵剂,本文以实验室前期筛选的63株乳酸菌作为研究对象,利用双层显色平板试验,氧化酶试验和高效液相色谱检测对产生物胺氧化酶菌进行筛选,并对所筛选的菌株做抑菌试验和耐盐耐亚硝酸盐等试验。结果表明:本次实验筛选出4株(1株Pediococcus acidilactici,1株Pediococcus pentosaceus,2株Lactobacillus plantarum)具有较强生物胺氧化能力的乳酸菌,其中以乳酸片球菌(Pediococcus acidilactici)的生物胺降解能力最好,其对苯乙胺、色胺、组胺、腐胺、尸胺、酪胺、亚精胺和精胺的降解率分别为56.71%、51.40%、40.86%、28.12%、18.92%、24.59%、18.20%和9.68%。通过抑菌试验和耐盐耐亚硝酸盐等试验发现乳酸片球菌对致病性大肠杆菌,单增李斯特菌,金黄色葡萄球菌具有良好抑菌效果,且耐盐能力在6%以上,耐亚硝酸盐能力在0.15 g/kg以上,最适温度为37℃,最适p H为6.5。     

葡萄球菌对发酵肉制品中生物胺降解机制研究进展

近年来,发酵肉制品因具有营养、美味等优点而深受消费者追捧,但发酵肉中常会存在生物胺,引发人们对其安全性的关注。生物胺是一类具有生物活性的低分子含氮有机化合物,适量剂量范围的生物胺有助于人体生长、增强代谢,过量则可能导致产生过敏、腹泻、致畸甚至死亡等不良反应。本文综述葡萄球菌降低发酵肉中生物胺含量的3 种机制：生物胺氧化酶氧化、菌株产生葡萄球菌素以及与其他菌株协同作用来控制发酵肉制品中生物胺含量。通过利用葡萄球菌降胺作用,为食品中生物胺的防控与消除提供思路。     

一种改进的产生物胺乳酸菌的检测方法

本文自行设计了一种对产生物胺(BA)乳酸菌进行检测的双层培养基法，利用该方法对实验室保藏菌种和一些发酵食品中的菌株进行了检测，实验证明此方法可在同一块平板上区分开产生物胺菌和不产生物胺菌，且方法简单、易于识别、准确性高，对生产和优良菌种发酵剂的开发很有帮助。     

发酵肉制品中细菌群落结构及其表皮葡萄球菌生物胺相关基因分析

该研究首先利用高通量测序技术对发酵肉制品中细菌群落结构进行分析;然后利用传统培养分离法及双层显色培养基对产生物胺的细菌进行分离筛选,并基于16S rRNA基因序列进行鉴定;最后利用聚合酶链式反应（PCR）对产生物胺细菌的赖氨酸脱羧酶、组氨酸脱羧酶和酪氨酸脱羧酶编码基因ldc、hdc、tdc进行扩增。结果表明,发酵肉制品中细菌主要为厚壁菌门（Firmicutes）的葡萄球菌属（Staphylococcus）。从发酵肉制品中共分离得到52株细菌,其中43株具有产生物胺能力,主要为表皮葡萄球菌（Staphylococcus epidermidis）;产生物胺能力较强的7株表皮葡萄球菌中均含有ldc和hdc基因,部分菌株检测到tdc基因,为评估发酵食品生物胺的潜在危害和快速检测奠定基础。     

一种改进的微球菌产生物胺的固体培养基检测法

自行设计了一种对产生物胺(BA)微球菌进行检测的固体培养基法,利用该方法对从自然发酵腊肉中分离出来的100株菌株进行了检测,同时以液体培养基法做对照,实验证明此方法可行,且该法简单,准确性高,对优良菌种发酵剂的开发很有帮助。     

传统发酵肉制品中降解生物胺菌株的筛选鉴定与应用研究

该研究利用高效液相色谱（HPLC）法对65株来源于传统发酵肉制品的耐盐、耐亚硝酸盐乳酸菌所产生物胺进行定性定量检测,筛选出降解率最高的不产生物胺菌株PL-ZL001。经形态观察、生理生化试验研究,并结合16S rDNA序列分析对其进行鉴定,同时探索其作为发酵剂对发酵香肠中生物胺含量的影响。结果表明,菌株PL-ZL001被鉴定为植物乳杆菌（Lactobacillus plantarum）,添加菌株PL-ZL001可抑制发酵香肠中6种生物胺的积累,尤其是对毒性最大的组胺含量的控制,效果显著优于商业用木糖葡糖球菌（Staphyloccus xylose）（P＜0.05）。     

黄酒酒曲中产生物胺细菌的分离鉴定

为探明黄酒酒曲中产生物胺细菌的菌相,深入研究黄酒中的生物胺形成机制,分别以4种黄酒酒曲(麦曲M、麦曲L、小曲Q、红曲H)为研究对象,采用脱羧酶培养基对产生物胺细菌进行初筛,高效液相色谱法复筛,并通过16S rDNA基因序列分析,对分离得到的产生物胺细菌进行鉴定。共筛选出42株产生物胺细菌,包括肠杆菌属(Enterobacter sp.)17株,乳杆菌属(Lactobacillus sp.)6株,克雷伯氏菌属(Klebsiella sp.)5株,沙门氏菌属(Salmonella sp.)4株,肠球菌属(Enterococcus sp.)3株,克罗诺菌属(Cronobacter sp.)3株,埃希菌属(Escherichia sp.)菌株2株,柠檬酸杆菌属(Citrobacter sp.)1株,葡萄球菌属(Staphylococcus sp.)1株。分离的产生物胺细菌中多数可同时产生多种生物胺,其中产腐胺、尸胺、组胺的菌最多,但未分离到色胺和亚精胺产生菌。     

Use of autochthonous Pediococcus acidilactici and Staphylococcus vitulus starter cultures in the production of "chorizo" in 2 different traditional industries

The present study determined how the different ripening conditions affected the growth and development of 3 autochthonous starter cultures, and the physico-chemical and sensory characteristics of chorizo. Each of 3 strains of Pediococcus acidilactici (MC184, MS198, and MS200) and one of Staphylococcus vitulus (RS34) were associated to prepare the starter cultures, P184S34, P198S34, and P200S34. Then, chorizo was prepared following 2 manufacturing procedures. The autochthonous starter cultures were able to compete and colonize the sausages in both ripening procedures. The use of the starter cultures showed evident differences by the texture analysis, with the control batches being generally tougher than the starter culture batches. Also, the highest biogenic amine (BA) levels were found in control batches and the lowest in P200S34 batches. While the use of these starter cultures does not change the sensory characteristics of these traditional fermented sausages, it improves their homogeneity and safety, except for P184S34 batch in which more BAs are detected in industry 2. PRACTICAL APPLICATION: The 3 autochthonous starter cultures selected could be used in traditional industries because they are able to compete well and colonize the dry fermented sausages "chorizo." The use of these starter cultures improves the texture and homogeneity of traditional fermented sausages. Biogenic amines decreased in the starter cultures batches improving the safety.     

Effects of starter cultures and nitrite levels on formation of biogenic amines in sucuk

The effects of starter cultures, starter A (Lactobacillus sakei+Staphylococcus carnosus) and starter B (Pediococcus acidilactici+Staphylococcus xylosus+Lactobacillus curvatus), nitrite levels (0, 75, and 150ppm) and ripening period on biogenic amine production were investigated in sucuk (Turkish dry-fermented sausage) production. Changes in biogenic amines, pH, water activity, moisture, non-protein nitrogen, nitrite and counts of lactic acid bacteria, Micrococcus/Staphylococcus, Enterobacteriaceae, and moulds-yeasts were determined. Ripening period had a very significant effect (P<0.01) on the all biogenic amines. Sausages produced by fermentation with starter cultures, as compared to natural fermentation (control), had lower amounts of putrescine, cadaverine and tyramine (P<0.05). Starter cultures A and B were found to have almost the same effect on the formation of biogenic amines. Use of nitrite in sucuk production affected the formation of biogenic amines except for spermidine and spermine (P<0.01). Lactic acid bacteria counts in the control group were lower than that of starter culture groups. Lactic acid bacteria, Micrococcus/Staphylococcus, and moulds-yeasts counts increased during ripening period while levels of species of Enterobacteriaceae decreased, which was also found to be under the detectable level (<100cfug(-1)), by day 3 of ripening.     

Safety improvement and preservation of typical sensory qualities of traditional dry fermented sausages using autochthonous starter cultures

Traditional dry fermented sausages are manufactured without addition of starter cultures in small-scale processing units, their fermentation relying on indigenous microflora. Characterisation and control of these specific bacteria are essential for the sensory quality and the safety of the sausages. The aim of this study was to develop an autochthonous starter culture that improves safety while preserving the typical sensory characteristics of traditional sausages. An autochthonous starter composed of Lactobacillus sakei, Staphylococcus equorum and Staphylococcus succinus isolated from a traditional fermented sausage was developed. These strains were tested for their susceptibility to antibiotics and their production of biogenic amines. This starter was evaluated in situ at the French traditional processing unit where the strains had been isolated. Effects of the autochthonous starter were assessed by analysing the microbial, physico-chemical, biochemical and sensory characteristics of the sausages. Inoculation with the chosen species was confirmed using known species-specific PCR assays for L. sakei and S. equorum and a species-specific PCR assay developed in this study for S. succinus. Strains were monitored by pulse-field gel electrophoresis typing. Addition of autochthonous microbial starter cultures improved safety compared with the traditional natural fermentation of sausages, by inhibiting the pathogen Listeria monocytogenes, decreasing the level of biogenic amines and by limiting fatty acid and cholesterol oxidation. Moreover, autochthonous starter did not affect the typical sensory quality of the traditional sausages. This is the first time to our knowledge that selection, development and validation in situ of autochthonous starter cultures have been carried out, and also the first time that S. equorum together with S. succinus have been used as starter cultures for meat fermentation. Use of autochthonous starter cultures is an effective tool for limiting the formation of unsafe compounds in traditional sausage while preserving their original and specific sensory quality.     

不同发酵方式对发酵马肉肠贮藏期品质劣变及食用安全性的影响

为有效确保发酵马肉肠贮藏期间的品质及安全性,研究添加汉逊德巴利酵母菌1808（Debaryomyces hansenii 1808）、木糖葡萄球菌21445（Staphylococcus xylose 21445）、植物乳杆菌E11（Lactobacillus plantarum E11）的复合发酵剂发酵组（FP）和单一乳酸菌发酵组（DZ）、自然发酵组（ZR）马肉肠贮藏过程中生物胺的变化,考察不同发酵方式对发酵马肉肠pH值、水分活度（aw）、色差、总挥发性盐基氮（total volatile basic nitrogen,TVB-N）含量、硫代巴比妥酸反应物（thiobarbituric acid reactive substances,TBARs）值及菌落总数的影响。结果表明：在贮藏期间,相比于自然发酵组和单一乳酸菌发酵组,添加复配发酵剂可有效减缓发酵马肉肠TVB-N含量、TBARs值、pH值的增幅,抑制贮藏后期有害杂菌的生长,防止产品过早发生腐败变质;此外,复配发酵剂可显著降低贮藏过程中马肉肠的水分损失（P＜0.05）,维持色泽的稳定,抑制生物胺（尸胺、腐胺、酪胺、组胺）的积累。综上,随着贮藏时间的延长,复配发酵剂可显著抑制发酵马肉肠品质的劣变及不良生物胺的过多积累。     

不同发酵肉中优良葡萄球菌的筛选鉴定

为获得适用于发酵香肠的优势葡萄球菌,本研究从国内外6种具有代表性的不同来源的发酵肉样品中分离到120株葡萄球菌.按照肉用发酵剂的基本原则进行初筛后共得到10株性能优良菌株.对这些菌株的蛋白酶、脂肪酶、硝酸还原酶和过氧化氢酶活性以及其在高盐和亚硝酸盐环境中的生长情况进行测定.发现菌株RG18和SL4有较高的蛋白酶、脂肪酶...     

Mixed starter cultures to control biogenic amine production in dry fermented sausages

Several combinations of an amine-negative Lactobacillus sakei strain, along with proteolytic Staphylococcus carnosus or Staphylococcus xylosus strains, were used to study the influence of mixed starter cultures on biogenic amine production during the manufacture of dry fermented sausages. Changes in pH, water content, proteolysis, microbial counts, and biogenic amine contents were simultaneously examined in a spontaneously fermented batch and in three mixed starter-mediated batches. A double-controlled microbial charge initially inoculated as mixed starter culture of L. sakei and Staphylococcus spp. (all amine-negative strains) drastically reduced tyramine, cadaverine, and putrescine accumulation. No production of other aromatic amines such as histamine, phenylethylamine, or tryptamine was observed in any batch. The polyamines, spermine and spermidine, were found in raw materials and their levels decreased slightly in the spontaneously fermented batch. No correlation between proteolysis and biogenic amine production was observed. The use of proper technological conditions favoring starter development and the use of the raw materials with good hygienic quality make it possible to produce fermented sausages nearly free of biogenic amines.     

Limosilactobacillus fermentum FSCBAD033控制带鱼加工下脚料发酵鱼露中生物胺含量研究

目的 控制带鱼加工下脚料低盐发酵鱼露中的生物胺含量。方法 以耐盐性高效生物胺降解乳酸菌Limosilactobacillus fermentum FSCBAD033为功能发酵剂控制带鱼加工下脚料发酵鱼露中的生物胺含量,并研究其对鱼露发酵过程中的pH值、NaCl含量、总可溶性氮(Total Soluble Nitrogen,TSN)含量、氨基酸态氮(Amino Acid Nitrogen,AAN)含量、挥发性盐基氮(Total Volatile Basic Nitrogen,TVB-N)含量以及发酵结束时感官风味的影响。结果 与Control组相比,接种L. fermentum FSCBAD033发酵剂对鱼露发酵过程中的NaCl含量影响不大(p>0.05),但可促进pH值下降以及TSN和AAN含量上升,并显著抑制TVB-N的产生(p<0.05)；显著降低发酵结束时鱼露中52.38%尸胺、40.21%组胺、45.44%酪胺、23.74%腐胺、52.67%苯乙胺和43.26%总生物胺(p<0.05),对含量较低的色胺、精胺和亚精胺影响不大；此外,接种L. fermentum FSCBAD033发酵剂可增强发酵结束时鱼露的鲜味和肉味,减弱氨味、腥味和臭味,整体提升鱼露感官风味。结论 L. fermentum FSCBAD033可作为带鱼加工下脚料低盐发酵的功能发酵剂,用于生产低盐、营养丰富、感官风味好且生物胺含量低的高品质鱼露产品。     

猪肉自然发酵香肠中细菌和乳酸菌对生物胺积累的作用

在猪肉香肠的自然发酵时，检测了选择的污染菌对形成的生物胺的性质和产量的作用。本文主要研究的是在肉腐败过程中胺的变化和微生物生长的变化，而且重点研究的是加入产酪胺的短乳杆菌（L．brevis）和产尸胺的阴沟肠杆菌（E．cloacae）的猪肉发酵香肠在发酵时生物胺的积累。在腐败和香肠发酵时相关微生物和胺的量较多。尸胺的高度积累和大量的不理想肠细菌有关，在香肠发酵时，酪胺的产生并不完全是乳酸菌的作用。另一方面，也涉及到必要的生产香肠的自然发酵菌，污染菌对于腐胺和其它生物胺的作用，现在还不清楚。